Subscriber access provided by Vanderbilt Libraries
Article
Synthesis of 1,2,3-Thiadizole and Thiazole Based Strobilurins as Potent Fungicide Candidates Lai Chen, Yu-Jie Zhu, Zhijin Fan, Xiao-Feng Guo, Zhi-Ming Zhang, Jing-Hua Xu, YingQi Song, Yury Yurievich Morzherin, Nataliya P. Belskaya, and Vasiliy A. Bakulev J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05128 • Publication Date (Web): 05 Jan 2017 Downloaded from http://pubs.acs.org on January 8, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 32
Journal of Agricultural and Food Chemistry
1
Synthesis of 1,2,3-Thiadiazole and Thiazole Based
2
Strobilurins as Potent Fungicide Candidates
3 4
Lai Chen, † Yu-Jie Zhu, † Zhi-Jin Fan,*, †, ‡ Xiao-Feng Guo, † Zhi-Ming Zhang, †
5
Jing-Hua Xu, † Ying-Qi Song, † Morzherin Y. Yurievich, § Nataliya P. Belskaya, §
6
Vasiliy A. Bakulev*, §
7 8 9 10
†
P. R. China
11 12
‡
15
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, P. R. China.
13 14
State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin 300071,
§
The Ural Federal University Named after the First President of Russia B. N. Yeltsin, Yeltsin UrFU 620002, Ekaterinburg, Russia
16 17
* Address correspondence to this author at State Key Laboratory of Elemento-Organic
18
Chemistry, Nankai University, No. 94, Weijin Road, Nankai District, Tianjin 300071,
19
P. R. China (telephone +86-13920714666; Fax:+86 22-23503620; e-mail:
20
[email protected], or
[email protected])
21
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
22 23
Abstract Strobilurin fungicides play a crucial role in protecting plants against different
24
pathogens and securing food supplies.
A series of 1,2,3-thiadiazole and thiazole
25
based strobilurins were rationally designed, synthesized, characterized and tested
26
against various fungi.
27
fungicidal activity of the target molecules.
28
a relative broad-spectrum of fungicidal activity.
29
activities against Gibberella zeae, Sclerotinia sclerotiorum and Rhizoctonia cerealis
30
with the median effective concentration (EC50) of 2.68, 0.44 and 0.01 µg/mL
31
respectively; it was much more active than positive controls enestroburin,
32
kresoxim-methyl and azoxystrobin with EC50 between 0.06 and 15.12 µg/mL.
33
Comparable or better fungicidal efficacy of compound 8a than azoxystrobin and
34
trifloxystrobin against Sphaerotheca fuliginea and Pseudoperonspera cubensis was
35
validated in the cucumber fields at the same application dosages. Therefore,
36
compound 8a was a promising fungicidal candidate worthy of further development.
Introduction of 1,2,3-thiadiazole greatly improved the Compounds 8a, 8c, 8d and 10i exhibited Compound 8a showed excellent
37 38
Keyword: Fungicide, strobilurin, 1,2,3-thiadiazole, thiazole, antifungal activity.
39
ACS Paragon Plus Environment
Page 2 of 32
Page 3 of 32
Journal of Agricultural and Food Chemistry
40 41
Introduction Plant pathogens can not only cause dramatic crop yield losses, but also greatly
42
affect the food security.1
43
loss for American corn growers was over one billion US dollars directly from
44
southern corn leaf blight which is caused by Cochliobolus heterostrophus, anamorph
45
Bipolaris maydis.2
46
mycotoxins called polyketides which contaminate grain based foods, causing a serious
47
food safety and economic problem in the United States.3
48
measures for managing plant pathogens are in great demand.
One extreme example is that estimated annually economic
Some plant pathogens such as the scab fungus could produce
Therefore, effective
49
Fungicides are an effective tool for plant disease management,4, 5 especially, new
50
fungicides are always welcome for farmers to phytopathogen managements. As the
51
second biggest classes of fungicides,6 being developed based on the lead compound
52
first isolated from fermentations of Stroblurus tenacellus in 1977 by block electron
53
transfer between cytochrome b and cytochrome c1,7 strobilurin fungicides have been
54
widely used against major plant pathogens.8
55
possesses a similar scaffold called β-methoxyacrylate.
56
fungicides have been launched and more are possible in the future.9-18
57
Majority of strobilurin fungicides Over a dozen strobilurin
Heterocyclic rings as a substructure is one of the important measures for novel
58
pesticide discovery including fungicides development.
59
pharmaceuticals and agrochemicals contain at least one heterocyclic ring.19
60
Heterocyclic rings functionalities differ from each other in biologically active
61
compounds,20 and work as scaffolds in active ingredients, prodrugs, tools for
ACS Paragon Plus Environment
Approximately 70% of
Journal of Agricultural and Food Chemistry
Page 4 of 32
62
fine-tuning physicochemical properties and isosteric replacements of functional
63
groups, alicyclic rings or other heterocyclic rings.21
64
derivatives possess properties that include environmental compatibility and broad
65
spectrum of bioactivities such as antifungal,22 antimicrobial,23 systemic acquired
66
resistance24 and antitumor activities.25
Thiadiazole and thiazole
67
This study was aimed at developing novel strobilurin fungicides by employing
68
the active scaffold of strobilurin fungicides and active 1,2,3-thiadiazole, thiazole or
69
thiazole’s derivatives as a substructures to the target molecules (Figure 1).
70
of novel 1,2,3-thiadiazole and thiazole based strobilurins were rationally designed,
71
synthesized and characterized.
72
candidate against Sphaerotheca fuliginea and Pseudoperonspora cubensis in the
73
cucumber fields.
A series
The active compound was evaluated as fungicide
74 75 76
Materials and Methods Equipment and materials: Melting points of synthesized compounds were 1
H and
13
77
taken on an X-4 melting point apparatus and were uncorrected.
C NMR
78
spectra were obtained on a Bruker Avance 400 MHZ spectrometer at 400 MHz and
79
100MHz in deutero-chloroform (CDCl3) and tetramethylsilane (TMS) as an internal
80
standard.
81
FTICR-MS instrument.
82
elemental analysis instrument.
83
1000CCD diffraction meter.
High resolution mass spectra (HRMS) were determined on a 7.0T Elemental analyses were performed on a Vario EL III Crystal structure was recorded by a Bruker SMART
All solvents were of analytical grade.
ACS Paragon Plus Environment
Page 5 of 32
Journal of Agricultural and Food Chemistry
General Synthetic Procedure for Compounds 3a-c (Figure 2).
84
Compounds
85
1a and 1b were commercially available.
86
the published procedures.26 Compound 2 was prepared from the corresponding acid
87
1.27
88
mL, 18.66 mmol) was added dropwise to a solution of 2 (12.44 mmol) in anhydrous
89
tetrahydrofuran (THF) (45 mL) at -30 °C under N2. The mixture was then stirred at
90
-30 °C for 1 h and at room temperature for another 1 h.
91
complete as monitored with thin layer chromatography (TLC), it was quenched with
92
the sat. aq ammonium chloride (NH4Cl) solution (50 mL).
93
and THF under vacuo, the aqueous phase was extracted with ethyl acetate (3×50 mL).
94
The combined organic layers were washed with water (50 mL), saturated brine (50
95
mL), and dried over anhydrous sodium sulfate.
96
evaporated.
97
mm)eluted with a mixture of ethyl acetate and petroleum ether (60-90 °C fraction) at a
98
ratio of 1:5 (v/v) to obtain compounds 3a-c in a 90-93% yield (Figure 2).
99
Compound 1c was synthesized according to
A solution of methyl magnesium bromide in ethyl ether (Et2O) (3 mol/L, 6.22
When the reaction was
After removal of Et2O
After filtration, the solvent was
The residue was then purified on a silica gel column (203 mm x 26
General Synthetic Procedure for Compounds 6a-c (Figure 2). Compound 4a
100
and 4b were commercially available.
Compound 4c was synthesized as reference
101
description.26
102
Dess-Martin periodinane (12.00 mmol) was added to a solution of compound 5 (10.87
103
mmol) in dichloromethane in an ice bath.
104
temperature overnight.
105
was then purified on a silica gel column eluted with a mixture of ethyl acetate and
Compound 5 was synthesized by reduction of compound 4.28
The reaction mixture was stirred at room
After filtration, the solvent was evaporated.
ACS Paragon Plus Environment
The residue
Journal of Agricultural and Food Chemistry
Page 6 of 32
106
petroleum ether (60-90 °C fraction) at a ratio of 1:4(v/v) to obtain compounds 6a-c in
107
a 50-80% yield.
108
General Synthetic Procedure for Compounds 8a-f (Figure 3). Compound 7 was
109
prepared according to Strazzolini and Pavsler.29
110
mmol) in 15 mL ethanol was added to the compound 3 or 6 (1.02 mmol) in ethanol
111
(15 mL), followed by addition of a catalytic amount of hydrochloric acid (2 mol/L,
112
0.1 mL.
113
removal of ethanol under vacuum, the residue was purified by recrystallization in
114
ethanol or silica gel column chromatography eluted with a mixture of ethyl acetate
115
and petroleum ether (60-90 °C fraction) at a ratio of 1:9-1:4 (v/v) to obtain the
116
corresponding compound 8 (Figure 3).
117
elemental analyses of the target compounds 8a-f were as follows:
118 119 120 121 122 123 124 125 126 127
A solution of compound 7 (1.12
The reaction mixture was stirred at room temperature overnight.
After
The yields, physical properties and HRMS or
Data for 8a: yellow solid; yield, 48%; m.p.: 70-72 oC; Anal. calcd for C16H18N4O4S: C, 53.03; H, 5.01, N, 15.46. Found C, 53.29; H, 4.86, N, 15.40. Data for 8b: yellow solid; yield, 67%; m.p.: 98-100 oC; HRMS (m/z) calcd for C15H16N4O4S: (M+H)+: 349.0892, found: 349.0969. Data for 8c: colorless crystal; yield, 53%; m.p.: 82-83 oC; Anal. calcd for C16H18N4O4S: C, 53.03; H,5.01, N, 15.46. Found C, 53.02; H, 4.99; N, 15.60. Data for 8d: white solid; yield, 70%; m.p.: 82-84
o
C; Anal. calcd for
C15H16N4O4S: C, 51.71; H, 4.63; N, 16.08. Found C, 51.83; H, 4.58; N, 16.23. Data for 8e: white solid; yield, 48%; m.p.: 91-93
o
C; Anal. calcd for
C26H34N4O6S: C, 58.85; H, 6.46; N, 10.56. Found C, 58.25; H, 6.26; N, 10.16.
ACS Paragon Plus Environment
Page 7 of 32
Journal of Agricultural and Food Chemistry
128 129
Data for 8f: yellow oil; yield, 47%; HRMS (m/z) calcd for C25H32N4O6S (M+H)+: 517.2043, found: 517.2114.
130
General Synthetic Procedure for Compounds 10a-o. Compound 9 was
131
prepared according to the report of Domagala et al.30 Compounds 9 (0.75 mmol),
132
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (0.17 g, 0.90
133
mmol) and 1-hydroxybenzotriazole (HOBt) (0.11 g, 0.77 mmol) were dissolved in
134
dichloromethane (25 mL) in an ice bath and was slowly warmed up to room
135
temperature and stirred for another 45 min.
136
(25 mL) was then added followed by addition of trimethylamine (Et3N) (0.09 g, 0.90
137
mmol), the reaction mixture was stirred at room temperature overnight, the organic
138
layer was successively washed with water (2×30 mL), saturated brine (40 mL), and
139
dried over magnesium sulfate and concentrated in vacuo.
140
residue were then purified on a silica gel eluted with a mixture of ethyl acetate and
141
petroleum ether (60-90 °C fraction) at a ratio of 1:1-1:6(v/v) (Figure 4). The yields,
142
physical properties and HRMS, elemental analyses of the target compounds 10a-o
143
were as follows:
144 145 146 147 148 149
A solution of amine in dichloromethane
Compound 10a-o in the
Data for 10a: yellow solid; yield, 83%; m.p.: 70-72 oC; HRMS (m/z) calcd for C17H19F2N5O3S (M+H)+: 412.1177, found: 412.1255. Data for 10b: yellow powder; yield, 89%; m.p.: 110-112 oC; HRMS (m/z) calcd for C18H19N5O3S (M+H)+: 386.1209, found: 386.1285. Data for 10c: yellow powder; yield, 69%; m.p.: 83-85 oC; Anal. calcd for C18H21N5O3S: C, 55.80; H, 5.46, N, 18.08. Found C, 55.83; H, 5.53; N, 18.23.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171
Data for 10d: colorless crystal; yield, 68%; m.p.: 71-73 oC; HRMS (m/z) calcd for C16H19N5O3S (M+H)+: 362.1209, found: 362.1289. Data for 10e: yellow oil; yield, 68%; HRMS (m/z) calcd for C20H27N5O3S2 (M+H)+: 450.1555, found: 350.1635. Data for 10f: white solid; yield, 57%; m.p.: 98-101 oC; HRMS (m/z) calcd for C17H19F2N5O3S (M+H)+: 412.1177, found: 412.1257. Data for 10g: colorless crystal; yield, 85%; m.p.: 131-133 oC; HRMS (m/z) calcd for C18H19N5O3S (M+H)+: 386.1209, found: 386.1287. Data for 10h: yellow crystal; yield, 90%; m.p.: 125-127 oC; Anal. calcd for C18H21N5O3S: C, 55.80; H,5.46, N, 18.08. Found C, 55.39; H, 5.53; N, 18.09. Data for 10i: white powder; yield, 82%; m.p.: 123-124 oC; Anal. calcd for C16H19N5O3S: C, 53.17; H, 5.30, N, 19.38. Found C, 52.47; H, 5.43; N, 19.67. Data for 10j: yellow oil; yield, 78%; HRMS (m/z) calcd for C20H27N5O3S2 (M+H)+: 450.1555, found: 450.1632. Data for 10k: yellow oil; yield, 87%; HRMS (m/z) calcd for C27H35F2N 5O5S (M+H)+: 580.2327, found: 580.2393. Data for 10l: yellow oil; yield, 91%; Anal. calcd for C28H35N5O5S: C, 60.74; H, 6.37; N, 12.65. Found C, 60.12; H, 6.18; N, 12.10. Data for 10m: yellow oil; yield, 91%; HRMS (m/z) calcd for C28H37N 5O5S (M+H)+: 556.2515, found: 556.2594. Data for 10n: yellow solid; yield, 64%; m.p.: 101-103 oC; HRMS (m/z) calcd for C26H35N5O5S (M+H)+: 530.2359, found: 530.2442.
ACS Paragon Plus Environment
Page 8 of 32
Page 9 of 32
Journal of Agricultural and Food Chemistry
172
Data for 10o: yellow oil; yield, 96%; HRMS (m/z) calcd for C30H43N5O5S2
173
(M+H)+: 618.2706, found: 618.2791.
174
Crystal Structure Determination for Compound 8b
175
The crystal of compound 8b was obtained from a mixture of dichloromethane
176
and petroleum ether for structure validation (Figure 5).
177
compound 8b were recorded on a Bruker SMART 1000CCD diffraction meter
178
equipped with graphite-monochromatic Mo Kα radiation (λ = 0.71073 Ǻ).
179
9395 measured reflections; 3936 unique (Rint = 0.0308) in the range of 1.96°≤ θ ≤
180
27.96°(h, -10 to 10; k, -13 to 13; l, -15 to 15), of which 2775 had |Fo| > 2|Fo|.
181
methods were used for the structure solving by the SHELXS-97 program.
182
structure were refined anisotropically by full-matrix least-squares for all non-H atoms
183
to give the final R=0.0359 and wR=0.1010 (w=1/[σ2((Fo2)+(0.0651P)2+0.0000P],
184
where P=(Fo2+2Fc2)/3), (∆/σ)max=0.982 and S=1.029. The hydrogen atoms were
185
located according to theoretical models.
186
Fungicidal Activity
187
X-ray intensity data of
A total of
Direct The
The fungicidal activities against Alternaria solani (A. s), Botrytis cinerea (B. c),
188
Cercospora arachidicola (C. a), Gibberella zeae (G. z), Phytophthora infestans (Mont)
189
de Bary (P. i), Physalospora piricola (P. p), Pellicularia sasakii (P. s), Sclerotinia
190
sclerotiorum (S. s) and Rhizoctonia cerealis (R. c) were detected in vitro at 50µg/mL
191
according to a reported method by using azoxystrobin as a positive control.31
192
any active compound with growth inhibition over 90% at 50 µg/mL, their median
193
effective concentration (EC50) were determined according to the reference by using
ACS Paragon Plus Environment
For
Journal of Agricultural and Food Chemistry
194
enestroburin, kresoxim-methyl and azoxystrobin as positive controls.32
195
The preventive activities of the target compounds against Pseudoperonospora
196
cubensis, Erysiphe graminis, Puccinia sorghi Schw, Colletotrichum lagenarium were
197
conducted at 400 µg/mL by fungal spores inoculation.9
198
by mother solution with distilled water (containing 0.1% Tween 80), the mother
199
solution was prepared by dissolving each target compound (0.0111 g) in 0.5mL DMF.
200
Control plants were sprayed with water solution containing the same concentration of
201
DMF and Tween 80 in the test solutions.
202
compared with the control plants by checking complete disease control as 100 and no
203
disease control as 0.
204
Evaluation of Fungicidal Activity of Compound 8a in the Field
205
The test solution was diluted
The percentage of disease control was
Azoxystrobin was used as a positive control.
Due to high potency in laboratory and greenhouse, the fungicidal activity of
206
compound 8a was further evaluated in the field.
207
formulation was prepared.
208
cucumbers was conducted in Wuqing County, Tianjin, China.
209
suspension concentrate), trifloxystrobin (50% water dispersible granule) and
210
pyraclostrobin (350 g/L, emulsifiable concentrate) were used as positive controls.
211
An application rates of compound 8a and positive standards were 37.5 and 75 g ai/ha
212
for S. fuliginea and P. cubensis, respectively.
213
the evaluated as Eqn. 1:
214 215
An emulsifiable concentrate (8.88%)
Its field efficacy against S. fuliginea and P. cubensis on Azoxystrobin (250g/L,
Disease index (DI) was determined by
DI=Σ(A×B)×100/(C×9)
(Eqn. 1)
Where A: number of disease leaves; B: corresponding grade of A; C: total
ACS Paragon Plus Environment
Page 10 of 32
Page 11 of 32
Journal of Agricultural and Food Chemistry
216
number of investigation leaves.
217
Preventative efficacy was evaluated as Eqn. 2
218
Efficacy (%)=[1-CK0×PT1/(CK1×PT0)]×100%
(Eqn. 2)
219
Where CK0: DI of control group before water application; PT0: DI of treatment
220
group before compound application; CK1: DI of control group after water application;
221
PT1: DI of treatment group after compound application.
222
conducted by Duncan's Multiple Range Test (DMRT).
Data analysis was
223 224
Results and Discussion
225
Chemistry
226
Compounds 3 and 6 were obtained from the corresponding acid 1 and ester 4,
227
respectively (Figure 2).
228
between compound 1 and N,O-dimethylhydroxylamine hydrochloride.27
229
intermediate 2 was reacted with methyl magnesium bromide in anhydrous THF to
230
give compound 3 in 90-93% yield. After reduction of compound 4 by sodium
231
borohydride and subsequent Dess-Martin oxidation, compound 6 was obtained in
232
50-80% yield.
233
Intermediate 2 was prepared with yield 90-98% by reaction This
The compounds 8 were obtained by the reaction between the intermediate 7 and
234
the compounds 3 or 6 catalyzed by hydrochloric acid (Figure 3 ).29
235
10a-10o were prepared by a condensation reaction between the hydrolytic product 9
236
of compounds 8a, 8c or 8e and amine R3NH2 with a yield ranging from 64-96%
237
(Figure 4).
All structures were confirmed by 1H NMR,
ACS Paragon Plus Environment
13
The compounds
C NMR, HR-MS or
Journal of Agricultural and Food Chemistry
238
elemental analysis.
239
the active scaffold of strobilurin and 1,2,3-thiadiazoles or thiazoles into one molecule,
240
a series of compounds were successfully designed, synthesized and well
241
characterized.
242
Fungicidal activity
243
Therefore, to develop new strobilurin fungicides by integrating
The in vitro antifungal activities were assessed at 50 µg/mL and results are listed
244
in Table 1.
The E-isomer of compound 8a (E-8a) exhibited slightly better fungicidal
245
activities against A. solani, G. zeae, P. piricola, B. cinerea, S. sclerotiorum and R.
246
cerealis than compound 8a (Z-8a). Because the difference between E-8a and Z-8a
247
was not significant, and the two isomers might be interconverted under biological
248
conditions, we used mixtures in all the bioassays in the same way as commercialized
249
positive controls.
250
good activity against G. zeae, B. cinerea, S. sclerotiorum, R. cerealis and P. infestans.
251
Compound E-8a exhibited a similar inhibition to azoxystrobin with 100% activity
252
against G. zeae and S. sclerotiorum.
253
inhibition against G. zeae, R. cerealis and P. infestans to azoxystrobin, and it showed
254
better efficacy against B. cinerea with 100% of growth inhibition than azoxystrobin
255
(79%).
256
infestans with 100% growth inhibition to azoxystrobin.
257
10c, 10d, 10g and 10i also showed the similar activity against PI with 100% growth
258
inhibition to azoxystrobin, and compound 10i exhibited the same activity against G.
Compounds 8 series with a 1,2,3-thiadiazole ring at R1 showed
Compound 8c exhibited similar 100%
Compound 8d had similar activity against S. sclerotiorum, R. cerealis and P. In addition, compounds 10b,
ACS Paragon Plus Environment
Page 12 of 32
Page 13 of 32
Journal of Agricultural and Food Chemistry
259
zeae, R. cerealis and P. infestans as azoxystrobin, having totally inhibited all fungal
260
growth. Table 2 showed EC50 values of compounds 8 and 10 series.
261
Being much better
262
than the positive controls such as enestroburin, kresoxim-methyl and azoxystrobin,
263
the compound 8a showed excellent activities against G. zeae, S. sclerotiorum and R.
264
cerealis with EC50 values of 2.68, 0.44 and 0.01 µg/mL respectively, and compound
265
8a also had a similar EC90 against G. zeae as azoxystrobin, and much better activity
266
against S. sclerotiorum with EC90 more than 10 times than that of the positive controls
267
such as enestroburin, kresoxim-methyl and azoxystrobin.
268
against B. cinerea greater than 20 times than that of the three positive controls such as
269
enestroburin, kresoxim-methyl and azoxystrobin.
Compound 8c had an EC90
270
The in vivo protective activity of all target compounds against P. cubensis, E.
271
graminis, P. sorghi Schw, and C. lagenarium at 400 µg/mL were evaluated (Table 3).
272
It showed that only 8a and 8c displayed better activity against P. cubensis. than the
273
positive control azoxystrobin.
274
1,2,3-thiadiazole ring and methyl group on the side chain at R1 and R2 respectively
275
could
276
exhibited 100% growth inhibition against C. lagenarium, which were higher than
277
azoxystrobin (80%).
278
of activity against E. graminis as azoxystrobin.
This indicated that the introduction of a
increase activities against P. cubensis.
Compounds 10a, 10f and 10n
Compounds 8a, 8c-e, 10d, 10e and 10i exhibited the same level
279
Subsequently, compounds 8a and 8c with good in vivo fungicidal activity were
280
chosen for further experiments in the greenhouse at the concentration of 50 µg/mL,
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 14 of 32
281
12.5 µg/mL, 3.13 µg/mL, 0.78 µg/mL and 0.20 µg/mL.
Compound 8c exhibited
282
lower activity against P. cubensis, P. sorghi Schw and C. lagenarium, however, it
283
showed the same activity against E. graminis as trifloxystrobin and azoxystrobin.
284
Compound 8a exhibited 100% inhibition activity against E. graminis even at 0.20
285
µg/mL, which was equal to azoxystrobin (100% inhibition) and better than
286
trifloxystrobin (95% inhibition) and enestroburin (30% inhibition).
287
exhibited 60% inhibition against P. cubensis at 50 µg/mL, whereas trifloxystrobin
288
only showed 20% inhibition, and enestroburin had no inhibition under the same
289
conditions.
290
warranted further development.
Compound 8a
Therefore, compound 8a exhibited high in vivo fungicidal activity and
291
These results indicated that the introduction of a 1,2,3-thiadiazole ring and a
292
methyl group on the side chain at R1 and R2 respectively could increase fungicidal
293
activities.
294
Evaluation of Fungicidal Activity of Compound 8a under the Field Condition
295
The compound 8a was chosen for field trials in 2015 against S. fuliginea and P.
296
cubensis on cucumbers (Table 5).
297
against S. fuliginea (p nd > nd > > 24.10 18.08
EC50 (µg/mL) and EC90 (µg/mL) P. i P. s EC50 EC90 EC50 EC90 nd nd nd nd 1.41 4.89 nd nd 8.76 70.17 nd nd 7.38 > nd nd 16.66 > nd nd 1.88 > 6.36 99.30 nd nd 13.92 > 0.41 > nd nd 99.84 > 18.40 > 0.71 12.08 10.71 > 0.40 7.58 3.68 >
R. c EC50 0.01 0.27 17.75 nd nd nd nd 0.07 0.12 4.43 0.06
S. s EC90 EC50 c > 0.44 > nd > 53.84 nd nd nd nd nd nd nd nd 16.82 nd 32.54 2.17 145.97 15.12 > 4.04
B. c: Botrytis cinerea; G. z: Gibberella zeae; P. i: Phytophthora infestans (Mont) de Bary; P.
s: Pellicularia sasakii; R. c: Rhizoctonia cerealis; S. s: Sclerotinia sclerotiorum. bnd: not determined; c >: data more than 200 µg/mL;
ACS Paragon Plus Environment
EC90 6.89 nd > nd nd nd nd nd > 81.89 >
Page 23 of 32
Journal of Agricultural and Food Chemistry
Table 3. In Vivo Fungicidal Activity of Compounds 8a-f and 10a-o. Fungicidal activity (%) at 400 µg/mL Compd. P. cubensis E. graminis P. sorghi Schw C. lagenarium 100 100 100 85 8a 0 0 0 0 8b 90 100 85 70 8c 0 100 30 80 8d 80 100 80 75 8e 80 60 70 85 8f 0 0 80 100 10a 30 0 85 80 10b 0 0 60 80 10c 0 100 85 85 10d 0 100 60 80 10e 75 30 0 100 10f 0 0 30 80 10g 0 70 60 80 10h 60 100 0 80 10i 65 0 0 0 10j 75 30 50 0 10k 85 0 0 60 10l 0 0 0 80 10m 60 0 80 100 10n 40 0 0 0 10o 85 100 0 80 Azoxystrobin
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Table 4. Greenhouse Fungicidal Activity Validation Studies In Vivo. Fungicidal activity (%) C(µg/mL) P. cubensis E. graminis P. sorghi Schw C. lagenarium 0.2 0 100 0 40 8a 0.78 0 100 20 55 3.13 0 100 55 60 12.5 15 100 100 85 50 60 100 100 98 0.2 0 95 10 0 8c 0.78 0 100 25 0 3.13 0 100 40 0 12.5 0 100 75 0 50 0 100 98 30 0 95 95 65 Trifloxystrobin 0.2 0.78 0 100 98 85 3.13 0 100 100 98 12.5 0 100 100 98 50 20 100 100 100 0.2 0 30 30 65 Enestroburin 0.78 0 65 75 85 3.13 0 95 98 95 12.5 0 98 100 98 50 0 100 100 100 0.2 0 98 100 100 Azoxystrobin 0.78 0 100 100 100 3.13 0 100 100 100 12.5 0 100 100 100 50 0 100 100 100 Compd.
ACS Paragon Plus Environment
Page 24 of 32
Page 25 of 32
Journal of Agricultural and Food Chemistry
Table 5. Fungicidal Efficacy of Compound 8a in the Cucumber Field. DD c Field Disease Compd. Efficacy(%) 5% 1% 76.61±3.63 ad ce 37.5 1.83 8.23 8a EC S. Azoxystrobin SC 37.5 1.66 9.59 70.19±2.91 bd de fuliginea Trifloxystrobin WG 37.5 1.64 10.04 68.02±5.58 b d CK ndf 1.07 20.63 nd nd nd 75 3.70 5.78 77.52±2.15 a cde 8a EC Trifloxystrobin WG 75 3.73 7.24 72.02±1.61 b d P. 75 3.57 5.78 77.02±1.77 a cd cubensis Pyraclostrobin EC CK nd 3.58 24.96 nd nd nd a b c Base DI, disease index base. After DI, disease index after compound application. DD, distinct difference. da and b: distinct difference at 95% of confidence limits; ec, d and cd: distinct difference at 99% of confidence limits. fnd, not determined. Rate (g ai/ha)
Base DIa
After DIb
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
FIGURE CAPTIONS Figure 1. Design of the title compounds Figure 2. General synthetic procedure for compounds 3a-c and 6a-c. Figure 3. General synthetic procedure for compounds 8a-f. Figure 4. General synthetic procedure for compounds 10a-o. Figure 5. Crystal structure for compound 8b by X-ray diffraction determination.
ACS Paragon Plus Environment
Page 26 of 32
Page 27 of 32
Journal of Agricultural and Food Chemistry
Figure graphics
CH 3
N N
S
S Hetero aryl
β-methoxyacrylate O
N Boc N
N
β-methoxyacrylate
Ph
O
N
Hetero aryl R1
CH 3 O β-methoxyacrylate: H 3 CO
N
O OCH 3 H3 CO
N
1
Figure 1
ACS Paragon Plus Environment
1
N H
R3
Journal of Agricultural and Food Chemistry
a
Reagents and conditions: (i) Et3N, CH3NHOCH3 HCl, EDCI, DMAP, dry CH2Cl2,
overnight; (ii) CH3MgBr, dry THF, 0 °C to r.t.; (iii) NaBH4, CH3OH;(iv) Dess-Martin periodinane, CH2Cl2, overnight. Figure 2
ACS Paragon Plus Environment
Page 28 of 32
Page 29 of 32
Journal of Agricultural and Food Chemistry
Figure 3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 4
ACS Paragon Plus Environment
Page 30 of 32
Page 31 of 32
Journal of Agricultural and Food Chemistry
Figure 5
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Graphic for table of contents
Fungcide Cadidate
ACS Paragon Plus Environment
Page 32 of 32